CN101370928A - Cell culture method and automatic culture device using the method - Google Patents

Abstract

The culture method of the present invention, which comprises: the step of injecting the cells obtained from the human body and the culture medium into the culture container; (2) carrying out ventilation or culture in the culture container with the culture medium and the cells (3) after washing the cultured cells cultured in the culture vessel, leaving a part of the washed cells and recovering them from the culture vessel to a cell recovery container; (4) injecting a culture medium into the cultured cells remaining in the culture container in the step (3); and (5) repeatedly implementing the steps (2) and (4) steps.

Description

Cell culture method and automatic culture device using the method

technical field

The present invention relates to a cell culture method and a cell culture device for carrying out the method, and in particular to a cell culture method and an automatic cell culture device capable of obtaining a large amount of cultured cells without mixing (contaminating) cultured cells in a human body with bacteria.

Background technique

In recent years, a large number of studies on regenerative medicine for regenerating damaged tissues or organs with reduced functions have been reported. In regenerative medicine, a large number of cells that constitute human tissues or organs are required, and artificially cultured cultured cells can be used for the cells required for regenerative medicine.

As a conventional device related to cell culture, there are culturing devices that can control the temperature, gas concentration, etc. In addition, it is also an automatic culture device that can control the input of cultured tissue into the culture container, the supply and discharge of culture reagents, and further control the atmosphere in the culture chamber.

Patent Document 1: Japanese Patent No. 3021789

Patent Document 2: International Publication No. WO2005/059091

In the former of the above-mentioned conventional culture apparatuses, operations such as replacement of the medium in the incubator and subculture to adjust the cell density to obtain a large amount of cultured cells are all performed manually by a researcher or the like. In the case of this device, for example, for operations such as medium replacement or subculture, after the operator opens the door of the device, while taking care not to allow bacteria to enter the incubator, he lifts up the cover of the incubator such as a petri dish, Use a sterilized pipette and proceed with great care. In addition, in the case of repeated subculture, it is necessary to transplant the proliferated cultured cells to a plurality of incubators multiple times, so even a skilled person may cause contamination.

On the other hand, the automatic culture device, which is the latter among the above-mentioned conventional culture devices, can also control the input of cells, the supply and discharge of culture reagents, and the atmosphere in the culture chamber as described above. However, the method adopted is to take out the cells cultured in the culture device and automatically transport them out of the device in the state of being injected into the container, and it is considered that there is still a problem that should be solved in terms of contamination when taking out the cultured cells.

In addition, when it is desired to regenerate tissues or organs of the human body, a large amount of cultured cells is required, but in order to obtain a large amount of cultured cells, the culture apparatus needs to be enlarged, and the amount of cells taken out from the human body needs to be increased. However, if the incubator is enlarged, of course, the cultivation apparatus will also be enlarged and the installation space will increase. In addition, if the amount of cells taken out from the human body is increased, there is a problem of causing great damage to the human body.

Among them, there is Patent Document 3 as a document related to a subculture technique for mass-proliferating experimental cells such as Paramecium. Regarding the technique described in this patent document 3, a new medium is supplied to cultured cells cultured in a culture container filled with a medium, and a part of the previous medium and cultured cells are washed to dilute the cultured cells in the culture vessel. Cultured cells. However, in this technique, the cells washed in the culture medium are directly used for measurement, and it is not suitable for obtaining a large amount of pure cells for human regenerative medicine.

Patent Document 3: Japanese Patent Laid-Open No. 2004-208663

Contents of the invention

It is an object of the present invention to provide a cell culture method and an automatic cell culture apparatus capable of obtaining a large amount of cultured cells used in regenerative medicine of the human body in response to the above problems.

In order to solve the above-mentioned problems, the cultivation method of the present invention includes:

(1) The step of injecting the cells and the culture medium obtained from the human body into the culture container; (2) performing the ventilation and the replacement of the culture medium in the above culture container containing the culture medium and the above cells, and at the same time carrying out the cell a step of culturing; (3) after washing the cultured cells cultured in the culture container, leaving a part of the washed cells and recovering them from the culture container into a cell recovery container; (4) a step of injecting a medium into the cultured cells remaining in the culture container in the above step (3); and (5) repeatedly performing the steps of the above step (2) and step (4).

In addition, in order to solve the above-mentioned problems, the automatic culture device of the present invention includes: a reagent supply unit that separately stores various reagents necessary for culture; a culture unit that stores the cell culture containers; cultured cells; a supply and discharge unit for supplying cells to the culture vessel, supplying reagents from the reagent supply unit to the culture vessel, and discharging cultured cells from the culture vessel to the recovery unit; a control unit for The reagent supply operation to the above-mentioned culture container, the cell culture operation in the above-mentioned culture container, and the discharge operation of the cultured cells from the above-mentioned culture container are controlled. A mechanism for at least one cell culture step in the reagent supply step of the supply and discharge unit, and the discharge step of the cultured cells.

According to the present invention, a large amount of cultured cells used in regenerative medicine of the human body can be obtained.

Description of drawings

Fig. 1 is a block diagram showing the unit configuration of the automatic culture device of the present invention.

Fig. 2 is a block diagram showing a schematic configuration of an automatic culture device according to the first embodiment of the present invention.

Fig. 3 is a perspective view of a culture container according to the first embodiment of the present invention.

Fig. 4 is a detailed side view of the culture container shown in Fig. 3 .

Fig. 5 is a cross-sectional view showing a nozzle piping portion of a culture container.

Fig. 6 is a flowchart of the cultivation method of the present invention.

Fig. 7 is a block diagram showing a schematic configuration of an automatic culture device according to a second embodiment of the present invention.

Detailed ways

(first embodiment)

Hereinafter, the present invention will be described with reference to the accompanying drawings. First, a first embodiment of an automatic culture apparatus for carrying out the cell culture method of the present invention will be described.

Fig. 1 is a block diagram showing a schematic configuration of an automatic culture device of the present invention. In FIG. 1 , 200 is a reagent supply unit separately storing various reagents necessary for cell culture of human cells, and its interior is configured to keep the temperature in a predetermined temperature range lower than normal temperature so that the stored reagents do not deteriorate. Reference numeral 300 denotes a culture unit, in which a culture container is accommodated. 400 is a recovery unit, which is a unit for recovering cells cultured in the culture unit or used reagents. 500 is a supply and discharge unit for supplying and discharging reagents or cells between the reagent supply unit 200, the culture unit 300, and the recovery unit. 600 is an environmental protection unit, which controls the temperature of the reagent supplied from the reagent supply unit 200 to the incubator, the temperature in the culture unit 300, and the concentration of carbon dioxide gas (CO ₂ ). 700 is an observation unit for observing the state of cell culture in an incubator accommodated in the culture unit 300 , and includes a microscope, a moving device for the microscope, or a lighting device for illuminating the inside of the culture container. 800 is a central processing unit (CPU) that controls the recovery unit 400 , the supply and discharge unit 500 , the environmental protection unit 600 , and the observation unit 700 . 900 is an operation unit including an operator for inputting operation commands to the automatic culture device, a monitor for displaying images obtained by the above-mentioned observation unit 700 , and the like.

Next, the above-mentioned units will be described in detail with reference to FIG. 2 .

The reagent supply unit 200 has a storage box (not shown) having an openable and closable cover, and a plurality of (three in the illustrated example) reagent storage boxes arranged at predetermined positions via support members inside the storage box. Reagent bags 5, 7, 9 were used. The storage box has a heat insulating material for blocking the storage space of the reagent bag from the outside and maintaining the space for storing the reagent bag at a predetermined temperature in order to prevent deterioration of the reagents contained in the reagent bags 5 , 7 , and 9 . The reagent bags 5 , 7 , 9 are preferably infusion bags made of foldable, flexible materials that are not toxic to cells. Specifically, for example, a bag made of vinyl chloride or silicone rubber is preferably used. Among them, the reagent bags 5 , 7 , and 9 separately store reagents necessary for culture, in this example, a cleaning solution, a stripping agent, and a culture medium. Among them, the stripping agent contained in the reagent bag 7 is a reagent necessary when the culture object is adherent cells, and the stripping agent is not required when the culture object is free cells.

The culture unit 300 has an incubator 3 serving as a constant temperature bath, and a culture container 1 accommodated in the incubator 3 . The incubator 3 has an openable and closable lid like the reagent supply unit 200, and the inside can be sealed by closing the lid. In addition, the incubator 3 has an observation window formed of a transparent material on the bottom surface facing the culture container 1 , so that the culture container 1 can be observed from the outside of the incubator 3 . In addition, the incubator 3 is thermostatically controlled by a heater described later so that the interior has a predetermined temperature. For this reason, in order to increase the heating efficiency of the heater, it is preferable to cover the periphery except the above-mentioned observation window with a heat insulating material. The culture container 1 is a device for culturing cells taken out of a human body, and is formed by using a material that is not toxic to cells and has light transmission, such as polystyrene or polyethylene terephthalate. In addition, the culture container 1 maintains the inside of the container in a sealed state with respect to the outside space so that the cells inserted into the inside or the cells to be cultured are not contaminated with miscellaneous bacteria. For this reason, the container mounting portion of the tube mounted on the culture container 1 is sealed.

The recovery unit 400 includes: a plurality of (three in this example) cell recovery bags 11, 13, 15 for recovering cultured cells cultured in the culture container 1, and a waste liquid container for recovering used reagents. 65 , a known heat sealing device 66 for heat sealing the tubular injection part of the cell recovery bag 11 , 13 , 15 . Like the reagent bags 5, 7, and 9, the cell recovery bags 11, 13, and 15 are preferably infusion bags made of foldable, flexible materials that are not toxic to cells. Specifically, for example, a bag made of vinyl chloride or silicone rubber is preferably used. In addition, the size of each cell recovery bag 11 , 13 , 15 is preferably such that the storage capacity is such that the cells cultured in the culture container 1 in one culture can be accommodated. In addition, the number of cell recovery bags included in the recovery unit 400 is three in this example, but it is preferable that the number is at least the number for repeatedly repeatedly culturing the same tissue cells in the culture vessel 1 several times in succession.

The supply and discharge unit 500 has: a supply pump 35 for supplying reagents, cells, and air into the culture container 1, a discharge pump 61 for discharging cultured cells or used reagents from the culture container 1, forming a reagent bag 5, The liquid delivery pipes 23, 25, 27, 29 of the flow path between 7, 9 and the culture container 1 are connected to the above-mentioned liquid delivery pipe 29 and the gas pipe 38 of the filter 37 described later in the incubator 3, connected to the above-mentioned The liquid delivery pipe 29 and the cell injection pipe 39 of the liquid storage container 43 are connected to the filter 77 installed inside the incubator 3 and the gas exchange pipe 75 in the inner space of the culture container 1, and the sleeve joint provided on the above-mentioned piping system. Flow valves 17, 19, 21, 33, 40. The liquid storage container 43 stores cells taken out from a living body and suspended in a medium or the like, and the opening is covered with a flexible film 45 . The membrane 45 that becomes the cover of the liquid reservoir 43 is provided for the syringe 41 that punctures the cells in the above-mentioned suspended state. Therefore, it is preferable that the film 45 has such flexibility as to seal the puncture mark of the syringe 41 and has a predetermined thickness so as not to mix bacteria and the like into the liquid storage container 43 .

Furthermore, the supply and discharge unit 500 has: a discharge pump 61 for discharging cultured cells or used reagents in the culture container 1, forming a gap between the culture container 1 and the cell recovery bags 11, 13, 15 and the waste liquid container 65. The discharge pipes 53, 55, 57, 59 of the flow passages, and the sleeve throttle valves 47, 49, 51, 53 arranged on the discharge passages. In this embodiment, an electromagnetically driven on-off valve capable of crushing a flexible tube and closing a flow path is used as the sleeve throttle valve. In addition, the supply pump and the discharge pump use a so-called thinning pump that winds a flexible tube into a plurality of rolls arranged in a cylindrical shape and rotates the rolls to squeeze out the fluid in the tube.

The environmental protection unit 600 has: a heater 31 provided on a part of the liquid supply pipe 29 for allowing the reagent to flow from the reagent bags 19 and 21 containing the stripping agent and the culture medium to the culture container 1; The internal heater, temperature sensor, carbon dioxide gas (CO ₂ ) sensor, etc. In addition, a CO ₂ supply tube is connected to the incubator 3, and the CO ₂ supply tube is connected to a CO ₂ compression bottle. The heater 31 heats during the process of supplying the stripping agent and the culture medium to the culture container 1 . In addition, a heater and a temperature sensor provided in the incubator 3 maintain the temperature in the culture container 1 at a predetermined temperature. In addition, the CO ₂ sensor detects the CO ₂ concentration inside the incubator 3 .

The observation unit 700 has: a microscope 39 installed outside the observation window of the incubator 3 for observing the state of cell culture in the culture container 1; a light source for illumination ( not shown), and a microscope moving device (not shown) for moving the microscope 89 two-dimensionally in a plane parallel to the bottom surface of the culture vessel 1 . The microscope 89 includes a camera, such as a CCD camera, capable of outputting captured image information as electrical signals. Microscope Moving Device A moving device consisting of a combination of motors and linear motion guide devices is arranged in crossed positions, and the microscope can be moved to any position by separating or linking the motors.

The central processing unit 800 controls the recovery unit 400, the supply and discharge unit 500, the environmental protection unit 600, and the observation unit 700 according to the cultivation conditions input from the operation unit 900 described later, and is composed of a CPU. CPU 800 has a ROM storing software for controlling operations of devices described later.

The operation unit 900 includes a manipulator for inputting operation (cultivation) conditions of the automatic culture apparatus, and a monitor for displaying the operation conditions input from the manipulator or images captured by the microscope 89 . Here, the pumps, valves, motors, heaters, and other actuators incorporated in the above automatic culture apparatus are driven by the drive circuit, but descriptions thereof are omitted.

Next, the incubator 1 will be described in detail using FIGS. 3 and 4 .

The cross-sectional shape of the culture container 1 is not particularly limited, but a flange is formed on the upper opening of the round petri dish in this embodiment. Using the flat surface of the flange, the lid 67 is adhered by welding or the like, and the culture container 1 is hermetically sealed. In addition, nozzles 69 , 71 , and 73 are inserted into the side surfaces of the culture container 1 , the liquid delivery pipe 29 is connected to the nozzle 69 , the discharge pipe 59 is connected to the nozzle 71 , and the gas exchange pipe 75 is connected to the nozzle 73 . Among them, FIG. 5 is a diagram showing the connection state of the nozzle 69 and the liquid delivery tube 29 in more detail.

Next, the positional relationship between the tips of the nozzles 69 , 71 , and 73 and the bottom surface of the culture container 1 will be described. The tip of the nozzle 69 connected to the liquid feeding pipe 29 is located in the liquid layer (the position of the liquid level during culture) or in the air layer in the culture container 1 . In addition, since the tip of the nozzle 71 connected to the discharge pipe 59 needs to leave a part (a small amount) of the cultured cells in the culture container 1 for the next culture, it is set from The inner bottom surface of the culture container 1 has a position with a predetermined gap. In addition, the front end of the nozzle 73 is connected to the gas exchange pipe 75 to serve as a supply and discharge port for exchanging the gas in the culture container 1 and the gas in the incubator 3, so it is set at the liquid layer in the incubator 1. high position.

Next, the cell culture method of the present invention will be described in relation to the operation of the above-mentioned automatic culture apparatus. However, in the following description, the case where the culture target is an adhesive cell will be described as an example.

(preparation for start of cultivation)

Before the culture starts, the operator installs the cell recovery bags 11, 13, 15 in the recovery unit 400 of the automatic culture device, and sterilizes the automatic culture device by radiating X-rays or γ-rays. Among them, the aseptic state of the cell recovery bags 11, 13, and 15 can be ensured by irradiating the bags from the time of bag production to packing. In addition, after the above-mentioned sterilization process is completed, the operator attaches the reagent bags 5 , 7 , and 9 storing the cleaning solution, stripping agent, and culture medium to the reagent supply unit 200 . In addition, the operator injects the cell liquid in a suspended state, such as cells or a culture medium, into the liquid storage container 43 using the syringe 41 . When these tasks are finished, the operator turns on the power of the automatic culture device, and inputs a culture start command from the operation unit 900 . Then, according to the operation flow in FIG. 6 , the CPU 800 executes cell culture by controlling each unit.

(Step 1: Media injection)

When an instruction to start the culture is input, the CPU 800 first executes control for injecting the culture medium. That is, the CPU 800 outputs a signal for opening the sleeve throttle valve 21 and the sleeve throttle valve 33 of the supply and discharge unit 500 , and then outputs a signal for operating the supply pump 35 . As a result, the supply pump 35 is driven while opening the flow paths of the liquid feeding pipes 27 and 29 . When the supply pump 35 is driven, the medium stored in the reagent bag 9 is transported into the culture container 1 through the liquid supply tubes 27 and 29 . At this time, electric current is supplied to heater 31 according to an instruction from CPU 800 , whereby the culture medium is heated in the tube. When a predetermined amount of culture medium is transferred to the culture container 1 , the CPU 800 closes the cartridge throttle valve 21 while driving the supply pump 35 . Then, the air in the incubator 3 is drawn into the gas pipe 38 and the liquid supply pipe 29 through the filter 37 . When the culture medium remaining in the liquid feeding tube 29 is used up, the supply pump 35 is temporarily stopped and the sleeve throttle valve 33 is closed according to a command from the CPU 800 . After the cartridge throttle valve 21 is closed, air is supplied to the liquid supply pipe 29 , whereby the reagent (culture medium) remaining in the liquid supply pipe 29 is quickly conveyed to the culture container 1 . As a result, clogging of the liquid delivery tube 29 by the reagent is prevented.

(Step 2: Cell Solution Injection)

When the injection of the medium is completed, the injection control of the cell solution is performed by the CPU 800 . The cell fluid is prepared in advance and stored in the liquid storage container 43 by the operator in advance. When an instruction to open the sleeve throttle valve 40 and an instruction to drive the supply pump 35 are output from the CPU 800, the sleeve throttle valve will be opened. 40 is opened and the supply pump 35 is driven. Thus, the cell solution is poured into the culture container 1 from the liquid storage container 43 through the liquid delivery tube 39 . In addition, when the injection of the cell solution is completed, the cartridge throttle valve 40 is closed and the supply pump 35 is stopped by a command from the CPU 800 .

(Step 3: Start of cultivation)

When the injection of the cell solution is completed, the CPU 800 controls the concentration of CO ₂ inside the incubator 3 and the temperature inside the incubator 3, and simultaneously cultures the cells for a preset time. The preset time is, for example, about 3 days, but may be changed according to the type of cells, the growth rate of cells, and the like.

(Step 4: Air Delivery)

After the above-mentioned set time elapses, the inside of the culture container 1 is ventilated. The inside of the culture container 1 of the present embodiment is substantially airtight during cell culture, and it is necessary to ventilate the inside of the culture container 1 within an appropriate time period. Therefore, the CPU 800 outputs a command to open the sleeve throttle valve 33 and a command to drive the charge pump 35 . When the sleeve throttle valve 33 is opened and the supply pump 35 is driven, the air in the incubator 3 sucked by the filter 37 is supplied to the culture container 1 through the gas pipe 38 and the liquid delivery pipe 29 (arrow A direction) At the same time, the air in the culture container 1 is discharged into the incubator 3 (in the direction of arrow B) through the gas exchange tube 75 and the filter 77 . In the flow chart of FIG. 6 , air transport is performed once, but it may be performed any number of times, or continuously, as long as the time of medium replacement and cell recovery described later is excluded. In addition, the exchange of air in the culture container 1 can be such that the air in the incubator 3 is supplied from the filter 77 to the culture container 1 (arrow C direction), and the air in the incubator 3 is discharged from the filter 37 (arrow D direction). Air in culture vessel 1.

(Step 5: Medium Replacement)

After the ventilation in the culture container 1 is completed, the culture medium is replaced at intervals of a predetermined time. The purpose of medium replacement is to discharge the medium in the culture vessel 1 so as to eliminate the metabolites of the cells eluted in the medium, and inject new medium into the culture vessel 1 to supplement nutrients, and it needs to be carried out regularly . Usually, the operations from Step 3 (S3) to Step 5 (S5) in FIG. 6 are performed at intervals of 3 days to 7 days, and this time is preset or can be changed arbitrarily according to the state of cell proliferation. In order to discharge the medium, the CPU 800 outputs a command to open the cartridge throttle valve 63 and simultaneously outputs a command to drive the discharge pump 61 . As a result, the sleeve throttle valve 63 is opened, the discharge pump 61 is driven, and the culture medium containing metabolic substances of the cells is discharged from the culture container 1 to the waste liquid container 65 through the discharge pipes 59 and 58 . In addition, when the culture medium is discharged from the culture container 1, the sleeve throttle valve 63 is closed, and the discharge pump 61 is stopped.

When the culture medium of the culture medium is exhausted, a new medium is supplied from the reagent bag 9 to the culture container 1 . In order to supply the culture medium, the CPU 800 outputs a command to open the sleeve throttle valves 21 and 33 and at the same time outputs a command to drive the supply pump 35 . As a result, the culture medium in the reagent bag 9 is conveyed via the liquid supply tubes 27 and 29 , heated by the heater 31 , and a predetermined amount is poured into the culture container 1 . When this medium replacement is performed, CPU 800 determines whether or not the number of medium replacements has been set in advance. In addition, when the medium exchange has not reached the set number of times, the process returns to step 4 (S4), and the air transport in step 5 (S5) and the medium exchange in step 6 (S6) are repeated.

(Step 7: Air Delivery)

In addition, when the culture medium has been exchanged for a predetermined number of times, the final air transport in the above-mentioned step 7 (S4) is performed in the same manner as in the step 4 (S4). However, during the cell culture period from Step 3 (S3) to Step 7 (S7), the CO ₂ concentration management and temperature management in the incubator 3 are continuously performed by the CPU 800 . In addition, during the cultivation period, the operator appropriately operates the observation unit 700 to photograph the cultivation state in the culture container 1 and display the photographed image on the monitor. The operator checks the progress status of the cultivation by observing the image displayed on the monitor.

(Step 8: Determination of the number of times of cell recovery)

When the air supply in Step 7 (S7) is performed within a predetermined period of time, for example, within several days, the step of recovering the cultured cells is performed. In the recovery step of the cultured cells, first, a determination is made for a set number of cell recovery performed thereafter. In this embodiment, the device is set to recover cells three times. In addition, in this Step 8 (S8), if it is determined that the cultured cells have been recovered three times, the next step proceeds to the final cell recovery process of Step 13 (S13). Also, if the cultured cells are recovered once or twice, the next process proceeds to Step 9 (S9).

(Step 9: Determination of the number of times of cell recovery)

In Step 8 (S8), it is judged whether or not the number of cell collections has been collected twice. In addition, when the determination result is not twice but once, the next process proceeds to the cell recovery process of step 10 (S10), and in addition, when the determination result is two times, the next process proceeds to step 11 (S11) cell recovery procedure.

(Step 10: First Cell Recovery)

When it is judged that the cell recovery is the first time, the process of discharging the culture medium in the culture container 1, the process of injecting the cleaning solution stored in the reagent bag 5, and the subsequent discharge process are performed to wash the cultured cells. . Subsequently, a step of injecting the peeling agent stored in the reagent bag 7 into the culture container 1 is performed. When a detachment agent is injected into the culture container 1 and a predetermined time elapses, the cultured cells are detached from the culture container 1 . Next, according to the result of judging that the cell collection is the first time, the step of collecting the cultured cells detached from the bottom surface of the culture container 1 into the cell collection bag 11 is carried out.

Therefore, in order to discharge the culture medium, the CPU 800 first inputs a command to open the sleeve throttle valve 63 and a command to drive the discharge pump 61 in the same manner as the culture medium discharge described in step 5 (S5). Thus, the culture medium in the culture container 1 is discharged. When the discharge of the culture medium is completed, the sleeve throttle valve 63 is closed by a command from the CPU 800, and the discharge pump 61 is stopped. Next, the CPU 800 outputs a command to open the cartridge throttle valves 17 and 33 and simultaneously outputs a command to drive the supply pump 35 in order to inject the cleaning solution into the culture container 1 . As a result, the sleeve throttle valves 17 and 33 are opened, and the supply pump 35 is driven to inject the cleaning solution stored in the reagent bag 5 into the culture container 1 . When a necessary amount of cleaning solution is injected into the culture container 1, the supply pump 35 is stopped, and the sleeve throttle valves 17 and 33 are closed. After the washing of the cultured cells with the washing liquid is completed, the washing liquid is sent to the waste liquid container 65 as in the discharge of the medium described in step 5 . Also, when the cleaning liquid is discharged from the culture container 1, the CPU 800 outputs a command to open the sleeve throttle valves 19 and 33 and a command to drive the supply pump 35 to inject the stripping agent into the culture container 1 . As a result, the cartridge throttle valves 19 and 33 are opened, the supply pump 35 is driven, and the stripping agent stored in the reagent bag 7 is injected into the culture container 1 . When the injection of the stripping agent is completed, the supply pump 35 is stopped, and the sleeve throttle valves 19 and 33 are closed. In addition, the peeling agent floats the cultured cells attached to the bottom surface of the culture container 1 after a short period of time. The CPU 800 injects a culture medium into the culture container 1 when the cultured cells are peeled off from the bottom surface of the culture container 1 . Here, the injection of the medium is performed to weaken the action of the stripping agent that adversely affects the cells. Therefore, the CPU 800 outputs a command to open the sleeve throttle valves 21 and 33 and a command to drive the charge pump 35 . As a result, the sleeve throttle valves 21 and 33 are opened, the supply pump 35 is driven, and the culture medium is injected from the reagent bag 9 into the culture container 1 . When the injection of the culture medium is completed, the supply pump 35 is stopped, and the sleeve throttle valves 21 and 33 are closed.

When the medium is injected to weaken the effect of the stripping agent, the adverse effect of the stripping agent on the cultured cells is reduced, and on the contrary, the cultured cells become easily attached to the culture container 1 again, so it is necessary to recover the cultured cells quickly. Therefore, the CPU 800 immediately outputs a command to open the cartridge throttle valve (here, the first recovery, so the cartridge throttle valve 47 is opened) and a command to drive the discharge pump 61 immediately after the injection of the culture medium. Thus, the cultured cells in the culture container 1 are recovered by the cell recovery bag 11 through the discharge pipes 59 and 53 . If the cultured cells are not sucked into the discharge pipes 59 and 53 , the CPU 800 closes the cartridge throttle valve 47 and also stops the discharge pump 61 . Then, a command to thermocompression-bond the tube portion of the cell recovery bag 11 is output from the CPU 800 to the heat sealing device 66 . Thus, the tube portion of the cell recovery bag 11 is heat-sealed. Subsequently, the cell recovery bag 11 is disconnected from the device by the operator. The cultured cell storage bag disconnected from the device is kept in a cold storage.

Here, since a predetermined gap is provided between the tip of the nozzle 71 attached to the culture container 1 and the bottom surface of the culture container 1, a part of the cultured cells remain in the culture container 1 without being collected. The gist of the present invention is to obtain a large number of cultured cells by repeatedly culturing the cultured cells remaining or inevitably remaining in the culture container 1 .

(second cell culture)

Therefore, when the first cell recovery in step 10 ( S10 ) is completed, CPU 800 executes steps 3 ( S3 ) to 9 ( S9 ) again for the cells remaining in culture vessel 1 . Thus, the cells remaining in the culture container 1 are cultured. The CPU 800 judges how many times the cells are collected this time in steps 8 ( S8 ) and 9 ( S9 ). Here, the judgment result is the second time.

(Step 11: Second Cell Recovery)

When it is determined in step 9 (S9) that the cell recovery is the second time, in step 11 (S11), the second cell recovery is performed in the same procedure as the cell recovery in step 10 (S10). However, in the second cell recovery of step 11 (S11), corresponding to the determination result of step 9 (S9), according to the instruction of CPU 800, the cells discharged from the culture container 1 are recovered by the cell recovery bag 13, and the heat-sealed device 66 is the same as Step 10 ( S10 ) in that the tube part of the cell recovery bag 13 is thermocompression-bonded.

(third cell culture)

When the second cell recovery in step 11 (S11) is completed, CPU 800 repeatedly executes steps 3 (S3) to 9 (S9) on the cells remaining in culture container 1 again. Thus, the cells remaining in the culture container 1 are cultured. The CPU 800 judges how many times the cells are collected this time in steps 8 ( S8 ) and 9 ( S9 ). Here, the judgment result is the third time.

(Step 12: The third cell recovery)

When it is determined in step 9 (S9) that the cell recovery is the third time, in step 12 (S12), the third cell recovery is performed in the same procedure as the cell recovery in step 10 (S10). However, in the third cell recovery of step 12 (S12), corresponding to the determination result of step 9 (S9), according to the instruction of CPU 800, the cells discharged from the culture container 1 are recovered by the cell recovery bag 15, and the heat-sealed device 62 is the same as step 10 (S10) in that the tube part of the cell recovery bag 15 is thermocompression-bonded.

When the third recovery of the cultured cells is completed, the cell recovery bags 11 , 13 , 15 containing the recovered cultured cells are transported to the clinic, and the cultured cells are used for tissue regeneration of the human body.

As described above, according to the present embodiment, when the cells cultured in the culture container are recovered, some of them remain in the culture container, and the cells remaining in the culture container are cultured again, thereby allowing the cells to proliferate in large quantities. . In addition, in this embodiment, the cells remaining in the culture container are cultured again, so a small-sized culture container can be used. Comparing this with the case of transferring cultured cells once cultured in a culture container to other multiple culture containers for continuous culture as in conventional subculture, the miniaturization of the device was achieved. Furthermore, according to this embodiment, it is only necessary to take out tissue cells from the living body once, so damage to the living body can be reduced.

In addition, according to this embodiment, the cells taken out from the living body are poured into the liquid storage container with the filter through the syringe, so that the contamination of the cells and miscellaneous bacteria can be prevented when the cells are stored in the culture container. Furthermore, according to this embodiment, the cultured cells are automatically recovered into the cell recovery bag, and at the same time the cell recovery bag is heat-sealed to block the cell recovery bag from the external environment, and then taken out, so it is possible to prevent cells and miscellaneous bacteria during cell recovery. pollute.

Furthermore, according to this embodiment, when the reagent is supplied into the culture container, the air in the culture container is driven out of the air in the culture container corresponding to the amount of the supplied reagent from the nozzle communicating with the inside of the incubator, so The reagent can be injected into the culture vessel smoothly. In addition, similarly, when liquids such as reagents in the culture container are discharged, air is supplied into the culture container from the nozzle communicating with the inside of the incubator corresponding to the discharge amount of the discharged reagent or liquid containing cells, so that the liquid can be smoothly The liquid in the culture container is removed in a timely manner.

In addition, according to the present embodiment, since air is supplied and discharged to the culture container through a filter, a clean culture environment can be provided and maintained.

In this embodiment, an example in which cell culture and recovery are repeated three times is described as an example, but the present invention is not limited thereto. The number of repetitions of cell culture and recovery can be set arbitrarily as long as it is at least 2 times depending on the amount of cells required. In this case, the number of cell recovery bags and the number of sleeve throttle valves installed in the recovery unit may be changed according to the number of times the cultured cells are recovered. Among them, as long as a device can be installed to prevent cell and miscellaneous bacteria contamination during cell recovery, it is not necessary to pre-fill multiple cell recovery bags in the automatic culture device, and the method of filling cell recovery bags to the automatic culture device can be used if necessary.

In addition, in this embodiment, nozzles 69, 71, and 73 are provided on the side surfaces of the culture container as shown in FIG. 5, so it can also correspond to a modified example in which a plurality of culture containers are stacked and arranged in the incubator.

Furthermore, the culture device of this embodiment can culture cells substantially continuously, and can recover cells at predetermined time intervals. Therefore, when regenerative treatment is performed on a patient's organs or tissues, for example, in leukemia treatment using mesenchymal stem cells, the mesenchymal stem cells may be supplied several times at predetermined intervals.

As shown in the above-mentioned embodiment, the cell recovery bags 11, 13, 15 and the waste liquid container 65 are connected to the discharge pipeline of a discharge pump 61 by means of the sleeve throttle valves 47, 49, 51, 63 respectively. There may be cases where waste or recycled cells remain in the tubing. When these waste liquids or dead cells, and further, the metabolites of the cells remain in the recovery system pipeline, contamination will occur in the repeated recovery of the next cultured cells. It is necessary to prevent the occurrence of such contamination.

(second embodiment)

FIG. 7 shows an automatic culture device according to a second embodiment capable of preventing such contamination. As shown in FIG. 7 , the automatic culture device of this embodiment is different from that of the above-mentioned first embodiment in that the number of cell recovery bags 11, 13, 15 and waste liquid containers 65 (4 in total) ) corresponding nozzles 101, 103, 105, 107 to replace the nozzle 71 of the discharge system provided on the culture container 1 of the first embodiment. That is, the nozzles 101, 103, 105, and 107 are connected to the discharge pipes 119, 121, 123, and 125, respectively. These discharge pipes 119 , 121 , and 123 are connected to the cell collection bags 11 , 13 , and 15 , respectively, and the discharge pipe 125 is connected to the waste liquid container 65 . Further, in the middle of these discharge pipes 119 , 121 , 123 , 125 , sleeve throttle valves 47 , 49 , 51 , 63 and discharge pumps 111 , 113 , 115 , 117 are respectively connected as shown in the figure. Wherein, the sleeve throttle valves 47, 49, 51, 63 are respectively installed on the suction side of the discharge pump in the drawings, but they can also be installed on the discharge side of the discharge pump. Among them, even in this embodiment, the discharge pumps 111 , 113 , 115 , and 117 and the sleeve throttle valves 47 , 49 , 51 , and 63 are all operated according to the instructions of the CPU 800 .

The automatic culture device of the present invention configured as described above differs in operation when the liquid in the culture container 1 is discharged from the automatic culture device of the first embodiment. That is, when the waste liquid in the culture container 1 is discharged, the sleeve throttle valve 63 is opened, and the discharge pump 117 is driven. In addition, when collecting cells in the culture container 1 , at the time of the first cell collection, the sleeve throttle valve 47 is opened, and the discharge pump 111 is driven. In addition, during the second cell recovery, the sleeve throttle valve 49 is opened, and the discharge pump 113 is driven; during the third cell recovery, the sleeve throttle valve 51 is opened, and the discharge pump 115 is driven.

According to this embodiment, in addition to the effect of the first embodiment, the discharge pipes 119, 121, 123, and 125 are provided independently, so that the cells recovered after the second time do not mix waste liquid or metabolic substances of the cells. Therefore, clean cultured cells can be obtained.

The above-mentioned second embodiment is to prevent both the waste liquid and the metabolites of the cells remaining in the pipeline from being mixed into the cells recovered after the second time during the previous recovery. It is deformed by mixing into the recovered cells after the second time. Referring to the above-mentioned second embodiment, this embodiment is completed only by setting a waste liquid recovery system independently for the first embodiment.

Claims (19)

1. automatic culture apparatus is characterized in that having:

The reagent feed unit, it is separately taken in and cultivates necessary plurality of reagents;

Cultivate the unit, it takes in cell culture container;

Reclaim the unit, it is recovered in the culturing cell of cultivating in the described culture vessel;

Give row the unit, its carry out to the cell of described culture vessel supply with, reagent from described reagent feed unit to the supply of described culture vessel and culturing cell from described culture vessel to the unitary discharge of described recovery; And

Control unit, the reagent of the described culture vessel of its subtend is supplied with action, interior cell cultures action and the culturing cell of described culture vessel controlled from the discharging operation of described culture vessel, and the culturing cell that remains in the described culture vessel when having the recovery culturing cell comprises based on the cell cultures operation of the unitary reagent supply of the described row of giving operation and the discharge operation mechanism at least once of culturing cell.

2. automatic culture apparatus as claimed in claim 1 is characterized in that,

The described row unit of giving has: the supply that is used for optionally a kind of reagent being supplied to described culture vessel from a plurality of reagent bags that are arranged at described reagent feed unit with pipeline, be arranged at valve and supply pump on the described pipeline.

3. automatic culture apparatus as claimed in claim 1 is characterized in that,

In the reagent that is accommodated in described a plurality of reagent bags, contain substratum and scavenging solution.

4. automatic culture apparatus as claimed in claim 3 is characterized in that,

In the reagent that is accommodated in described a plurality of reagent bags, also contain stripper.

5. automatic culture apparatus as claimed in claim 1 is characterized in that,

The described row unit of giving has: reclaim culturing cell a plurality of collection bags, accommodate the reagent that has used waste fluid container, be used for discharge that the reagent that optionally will use from described culture vessel and culturing cell be discharged to described collection bag or waste fluid container with pipeline, be arranged on valve and overboard pump on the described pipeline.

6. automatic culture apparatus as claimed in claim 1 is characterized in that,

Described culture vessel is provided with the nozzle that a plurality of and described row of giving unit is connected.

7. automatic culture apparatus as claimed in claim 6 is characterized in that,

Be provided with specified gap between the bottom surface of the described front end in culture vessel of giving the described nozzle that the unitary discharge of row is connected with pipeline and described culture vessel, by described gap, when the recovery of culturing cell, the part of culturing cell residues in the culture vessel.

8. automatic culture apparatus as claimed in claim 5 is characterized in that,

Described collection bag is made of the material with thermo-compressed, has to be used for and the tube portion of described discharge with pipe connection.

9. automatic culture apparatus as claimed in claim 8 is characterized in that,

Described give the row unit have to by from the instruction of described control unit specific the tube portion of collection bag carry out the hot-sealing device of thermo-compressed.

10. automatic culture apparatus as claimed in claim 5 is characterized in that,

The discharge of described deliverying unit with pipeline by the single pipe that is connected to described culture vessel being connected with a plurality of pipes that are connected to a plurality of collection bags and waste fluid container and constituting, overboard pump is configured on the described single pipe, and valve is configured on each pipe that is connected to a plurality of collection bags and waste fluid container.

11. automatic culture apparatus as claimed in claim 5 is characterized in that,

The discharge of described deliverying unit has a plurality of pipes that connect described culture vessel, waste fluid container and a plurality of collection bags respectively with pipeline, disposes overboard pump and valve at each pipe.

12. automatic culture apparatus as claimed in claim 5 is characterized in that,

The discharge of described deliverying unit comprises with pipeline: the waste liquid that connects described culture vessel and waste fluid container is with pipe, single pipe that is connected with described culture vessel and a plurality of pipes that are connected this single pipe and a plurality of collection bags;

Described waste liquid with pipe with single pipe that described culture vessel is connected on dispose overboard pump and valve respectively.

13. a cultural method is characterized in that, comprising:

(1) cell that will obtain from human body and the substratum step that is injected into culture vessel;

(2) put into the ventilation of described culture vessel of substratum and described cell and the replacing of substratum, carry out the step of cell cultures simultaneously;

(3) after the culturing cell of cultivating cleans, the step of cell returnable will after described its part of the cell residue that has cleaned it be recovered in the described culture vessel in for described culture vessel;

(4), inject the step of substratum for the culturing cell that in described step (3), remains in the described culture vessel; With

(5) carry out the step of described step (2) and step (4) repeatedly.

14. cell culture processes as claimed in claim 13 is characterized in that,

Carry out described step (4) and step (5) at least once.

15. cell culture processes as claimed in claim 13 is characterized in that,

In the recycling step of the cell of described step (3), also be included in after the step that the culturing cell in the described culture vessel is cleaned, peel off the step of culturing cell from described culture vessel by injecting stripper.

16. cell culture processes as claimed in claim 15 is characterized in that,

After stripper in described step (3) injects, also be included as and weaken stripper injects new substratum in described culture vessel to the influence of culturing cell step.

17. cell culture processes as claimed in claim 13 is characterized in that,

The culturing cell that reclaims in described step (5) is reclaimed by the container different with the container of recovery cell in the step (3).

18. cell culture processes as claimed in claim 15 is characterized in that,

The culturing cell that reclaims in described step (5) is reclaimed by the container different with the container of recovery cell in the step (3).

19. cell culture processes as claimed in claim 16 is characterized in that,

The culturing cell that reclaims in described step (5) is reclaimed by the container different with the container of recovery cell in the step (3).

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